The aims of this research project are to understand the regulation of viral gene expression during herpesvirus infection, evaluate the feasibility of herpesvirus vectors as gene therapy vectors, and determine the herpesvirus genes that would be most effective for development as potential subunit vaccines. Understanding the regulation of HSV gene expression is essential for the development and evaluation of successful vaccines, particularly live virus vaccines. During herpes simplex virus infection, approximately 75 genes are expressed in a well ordered process that can be conveniently divided into three phases based on the temporal order of mRNA synthesis. Immediate-early genes are expressed soon after virus infection, and their gene products are required for the expression of early genes, many of whose gene products are involved in viral DNA replication. Expression of viral late genes requires viral DNA replication and functional immediate-early gene products. Currently, two genetic approaches are being used to identify the factors that regulate HSV temporal gene expression. In one approach, a temperature-sensitive cell line, ts13, that is defective for the functional eukaryotic transcription factor TAF250 at the non-permissive temperature is being used to determine the role of this factor in HSV transcription. While immediate-early genes are transcribed at normal levels, the transcription of early and late genes is reduced. Interestingly, even though some late gene transcription does occur, late proteins are not made in these cells at the non-permissive temperature. In a second approach, we are constructing recombinant viruses that require a genetically altered TATA-binding protein (TBP) for the expression of specific viral genes. Mutagenesis of this altered specificity TBP should be useful for determining the interactions of TBP with the transcriptional complexes at the different types of HSV promoters. A second part of this research project is designed to evaluate the feasibility of replication-incompetent herpes simplex viruses as gene therapy vectors. Studies during FY98 have focused on construction of recombinant vectors that can be induced to express foreign genes in the presence of the antibiotic tetracycline. These vectors will be evaluated in neuronal cell culture systems for their ability to express foreign genes in the presence of tetracycline. The third part of this research project is designed to evaluate various HSV gene products as potential components of subunit vaccines. Experiments are underway to evaluate the protective effect of multiple HSV genes delivered simultaneously by DNA immunization.